Modern agriculture aims to reduce continuous applications of synthetic chemical pesticides for pest control because of issues such as pollution, loss of trade and disruption of biological control agents, potentially leading to pest outbreaks. Botanical insecticides are a good alternative to synthetic pesticides, as they have lower environmental hazard, are biodegradable and can be used shortly before harvesting. We tested the effect of essential oils (EOs) and aqueous extracts (AEs) of Chenopodiaceae plants (Dysphania ambrosioides (=Chenopodium ambrosioides), Chenopodium album, C. berlandieri subsp. nuttalliae, C. graveolens and Beta vulgaris), incorporated into the diet under lab conditions, on a widely distributed insect pest in America, the cabbage pest, Copitarsia decolora Guenée 1852 (Lepidoptera: Noctuidae). The EO of C. graveolens at 0.5% reduced the larvae and pupae weight by 30 and 15%, respectively; increased the larval period length by 17% and reduced fecundity and fertility by 80 and 85%, respectively related to the control. EOs of C. berlandieri and B. vulgaris at 0.5% increased larval and pupal period length by 22% and 38% and both reduced fecundity and fertility by 99% related to the control. None of the plant extracts tested caused immediate C. decolora mortality, but EOs reduced mean survival time and reproductive capacity of the insect. This is the first report of C. berlandieri EO effect against an insect pest. The EOs of C. graveolens, B. vulgaris and C. berlandieri have great potential as alternatives to synthetic chemicals for insect control and deserve further exploration.
Botanical insecticides are a viable alternative to synthetic insecticides, and they degrade quickly, retard the development of insecticide-resistance, can be used shortly before harvest (Kühne 2008; Silva-Aguayo 2013) and are environment friendly. An example is UDA-245, a Chenopodium-based natural insecticide (Chiasson et al. 2004; Bostanian et al. 2005). Components of Chenopodiaceae species such as Dysphania ambrosioides (L.) Mosyakin & Clemants (formerly Chenopodium ambrosioides) have been reported to produce several effects in insects. They reduced larval weight and number of progeny of Sitotroga cereallela (Olivier; Lepidoptera: Gelechiidae) (Gemechu et al. 2013); repelled or killed Coleoptera Sitophilus zeamais (Motschulsky) (Chu et al. 2011) and a commercial product derived from D. ambrosioides killed the long tailed mealy bug Pseudococcus longispinus (Targioni & Tozzetti) (Cloyd & Chiasson 2007). Other species affected by this plant are lepidopterans (Pieris spp.), coleopterans (Popillia japonica (Newman), Leptinotarsa spp., Callosobruchus maculatus and Phyllobius spp.), and dipterans (Aedes spp. and Cochliomyia hominivorax (Coquerel)). Chenopodium graveolens ((Willd.) W. A. Weber) has been reported to be toxic to the lepidopteran Tuta absoluta, the hemipteran Bemisia tabaci (Gennadius) and the thysanopteran Frankliniella schultzei (Trybom) (Barbosa et al. 2011). Chenopodium album (L.) reduces larval growth, pupal weight and kills the larvae of the noctuid Mamestra configurata (Walker). The methanolic extract of foliar parts of Beta vulgaris L. (Caryophyllales: Amaranthaceae) (10 g/100 mL) repelled and reduced the fecundity in the spider mite Tetranychus cinnabarinus (Boisduval; Trombidiformes: Tetranychidae) (Mansour et al. 2004). Alkaloids, steroids, flavonoids, phenols, and saponins found in Chenopodiaceae plants has been identified and have been deemed to be responsible for various biological effects on insects (Ibrahim et al. 2007; Kokanova-Nedialkova et al. 2009).
We evaluated the effect of essential oils (EOs) and aqueous extracts (AEs) of 5 Chenopodiaceae plants: Chenopodium album (L.), D. ambrosioides, C. berlandieri subsp. nuttalliae ([Saff.] H. D. Wilson & Heiser), C. graveolens ([Willd.] W. A. Weber), and Beta vulgaris rubra (L.) on the development and reproductive potential of an important pest in North, Central and South America, i.e., Copitarsia decolora (Guenée) (Lepidoptera: Noctuidae). This moth is a pest of Brassicaceae species such as cabbage (Brassica oleracea var. capitata), cauliflower (Brassica oleracea var. botritys), broccoli (Brassica oleracea var. italica) (Suárez-Vargas et al. 2006), and other important economic plants like onion (Allium cepa L.), garlic (Allium sativum L.; Alliaceae), coriander (Coriandrum sativum L.; Apiaceae), potato (Solanum tuberosum L.; Solanaceae), strawberry (Fragaria spp.; Rosaceae), apple (Malus spp. Rosaceae), corn (Zea mays L.; Poaceae), asparagus (Asparagus officinalis L.; Liliaceae), spinach (Spinacia oleracea L.; Amaranthaceae) among others (Moreno & Serna 2006; Angulo & Olivares 2003, 2010). Cabbage shipments exported from Mexico to the USA are strictly monitored for the presence of C. decolora for quarantine purposes (USDA 2013). Control of C. decolora larvae in México is attempted by using various broad spectrum insecticides.
Materials and Methods
INSECT LABORATORY COLONY
We reared our insects according to Callado-Galindo et al. (2013) and separated genders according to Angulo & Olivares (2010). Adults (10–12 females and 6-8 males) were caged in 20 × 20 × 20 cm acrylic cages and 6-cm diam Petri dishes with cotton were used to dispense a 10% honey solution. The colony was maintained in a brood chamber at 20 ± 5 °C , 50 ± 5% RH and 12:12 h L:D.
ORIGIN OF PLANT MATERIAL
Chenopodium album was collected from Jumiltepec, Morelos State, C. graveolens from Juchitepec, México State and D. ambrosioides, C. berlandieri subsp. nuttalliae and B. vulgaris were bought from Cuautla local market, Morelos, México. Plants were identified by Dr. Rolando Ramírez at University of Morelos State Herbarium (HUMO) where specimens were deposited.
ESSENTIAL OILS AND AQUEOUS EXTRACTS
The whole plant of each of the 5 above-mentioned Chenopodiaceae species was used to obtain EOs and AEs. Essential oils were obtained by steam distillation according to Jardim et al. (2008). In brief, 4 L of chopped plant material was placed in a 12 L round-bottomed flask. Steam was passed through the material until no additional oily material was recovered. Water was removed from the solution by freezing and addition of anhydrous sodium sulfate. The resulting oil was kept at 4 °C in a brown glass container.
All plant material used for AEs was dried for 24 h at 50 °C in a laboratory stove, except for the beet root that was sliced (3–5 mm) and dried under a conventional fan oven for 4 d. The dried material was turned into a powder in an electric mill (IKA® WERKE, model MF 10 basic) and filtered through a no. 40 mesh sieve. Aqueous extracts (w/v) were obtained by soaking the dried powder in water in a 500 mL Erlenmeyer flask under constant stirring for 24 h at 150 rpm. The resultant extracts were filtered twice through gauze and was dried in an extraction hood before use.
Three bioassays were conducted; 2 for EOs and 1 for AEs. We tested 0.1 and 0.5% (v/v) EOs of C. album, C. graveolens, C. berlandieri subsp. nuttalliae, D. ambrosioides and B. vulgaris. Tween 20™ was added to EOs (1:5) to stabilize the solution prior blending it into the diet. Diet alone and diet with Tween 20 (called stabilizer from now on) were used as controls.
We tested 5% aqueous extracts (w/v) of C. album, C. graveolens, C. berlandieri var. nuttalliae, D. ambrosioides and B. vulgaris. Normal diet was used as the control. For all bioassays, 60 newly-hatched larvae were individually tested and reared separately in 30 mL transparent plastic containers for each treatment and kept under the same conditions. Diet was replaced twice weekly. For each treatment we recorded larval mortality, the weight of the larvae and pupae, the duration of the larval and pupal periods and adult fecundity, fertility and longevity.
EFFECT ON FECUNDITY AND FERTILITY
Male and female moths from the same treatment were allowed to mate in transparent acrylic boxes (20 × 20 × 20 cm). After mating, females were placed individually into plastic bags. According to Callado-Galindo et al. (2013) females oviposit more than 90% of their egg load by day 10 of the oviposition period, thus we collected eggs masses daily for 10 d and incubated the eggs under the same conditions as the colony on damp filter paper in 6-cm diam Petri dishes. Adults were fed a 10% honey solution from small-perforated Eppendorf tubes until they died. Fecundity was defined as the total number of eggs laid by a female and fertility as the number of eggs that produced larvae.
We used a one-way Analysis of Variance or Kruskal-Wallis procedure followed by a Holm-Šidák test, depending on the normality of the data. Mean survival time (from larval to adult death) were obtained from Kaplan-Meier curves followed by a Gehan-Breslow Test (Rich et al. 2010). All statistical analyses were conducted in SigmaPlot 11 and the rejection probability was set at 0.05. Reported values are mean ± SE unless otherwise stated.
LARVAL AND PUPAL WEIGHTS
Essential oils at 0.1% and 0.5% significantly reduced larval weight (H = 38.5, df =6, 400, p ≤ 0.001 and H = 67, df = 6, 205, p ≤ 0.001, respectively), in treatments in which larva were fed C. graveolens and B. vulgaris EOs at 0.1%. These treatments caused significant weight reductions of 15% and 14%, in relation to the control (Table 1). EOs of C. graveolens and D. ambrosioides at 0.5% significantly reduced the larval weight by 33% with respect to the control (Table 1). There was not a statistical difference between the aqueous control and the stabilizer. Larvae treated with AEs had similar weights (F = 2.1, df = 5, 328, p > 0.05; Table 1).
Effects of essential oils and aqueous extracts of Chenopodiaceae plant species on mean (± SE) Copitarsia decolora larval weights (mg).
EOs significantly affected pupal weights at both concentrations (Table 2) (H = 37.5, df = 6, 353, p ≤ 0.001 at 0.1% and H = 53.6, df = 6, 173, p ≤ 0.001 at 0.5%). At 0.5%, EOs of C. graveolens and B. vulgaris produced significantly lighter pupae than the control (15% and 12%, respectively). No AEs significantly reduced pupal weights in relation to the control but C. berlandieri significantly increased it albeit this increase was negligible (F = 3.4, df = 5, 259, p = 0.005; Table 2).
LARVAL AND PUPAL PERIOD LENGTH
All EOs at 0.1% and 0.5% increased the duration of the larval period in relation to the control (H = 60.9, df = 6, 400, p ≤ 0.001 and H = 92.6, df = 6, 205, p ≤ 0.001, respectively; Table 3). The largest increases at 0.1% were produced by D. ambrosioides (20%), C. berlandieri (13%) and C. graveolens (11%), respectively. At 0.5%, C. berlandieri (22%), D. ambrosioides and B. vulgaris (19%) produced the longest larval period. In both concentrations, these figures were not significantly different from those of the stabilizer treatment. No difference in duration of the larval period was observed with AEs (H = 5.4. df = 5, 331, p > 0.05, Table 3).
No significant difference was observed in the durations of the pupal period of insects treated with EOs at 0.1% (H = 7.9, df = 6, 353, p > 0.05; Table 4). However at 0.5% concentration, all EOs increased the length of the pupal period length in relation to the control (F = 74.917, df = 6, 172, p < 0.001; Table 4), there was a significant increase of 41% with C. album and 38% with C. berlandieri with respect to the control. A significant difference in the durations of the pupal period was observed in insects fed with AEs in that a significant 9% pupal period increase was observed for C. album and a 7% increase with both C berlandieri and D. ambrosioides with respect to the control (F = 3.4, df = 5, 259, p = 0.005; Table 4).
Effects of essential oils and aqueous extracts of Chenopodiaceae plant species on mean (± SE) Copitarsia decolora pupal weights (mg).
Effects of essential oils and aqueous extracts of Chenopodiaceae plant species on mean (± SE) Copitarsia decolora larval period (d).
FECUNDITY AND FERTILITY
In C. decolora fed with EOs of B. vulgaris, and C. graveolens at 0.1% there was a significant reduction in fecundity (82% and 69%, respectively) compared to the control (F = 22.7, df = 6, 101, p < 0.001; Table 5). Further significant reductions in fecundity were obtained with 0.5% EOs of B. vulgaris, C. berlandieri, D. ambrosioides and C. graveolens (99, 94, 88 and 80%, respectively) (F = 38.5, df = 6, 74, p < 0.001; Table 5). At 5% AEs of D. ambrosioides, C. berlandieri and B. vulgaris significantly reduced fecundity by 70, 58 and 51%, respectively (F = 14.4, df = 5, 97, p < 0.001; Table 5).
Essential oils of Beta vulgaris and C. graveolens at 0.1% significantly reduced the number of fertile eggs by 89% and 75%, respectively (F = 24.9, df = 6, 101, p < 0.001) (Table 6) and at 0.5% concentration, fertility was reduced by 99, 96, 93 and 85% by B. vulgaris, C. berlandieri, D. ambrosioides and C. graveolens respectively (F = 36.6, df = 6,74, p < 0.001). The largest reduction on fertility (75%) with AEs was caused by D. ambrosioides and C. berlandieri (F = 13.4, df = 5, 97, p < 0.001) (Table 6).
MEAN SURVIVAL TIME
Beta vulgaris essential oil at 0.1% produced a significantly shorter mean survival time (56.4 ± 3.1 d) than D. ambrosioides (65.9 ± 2.2 d). Days alive did not differ between control (58.7 ± 2.2 d) or the stabilizer (63.6 ± 2.6 d) treatment (Gehan-Breslow Test = 29.941, df = 6, p < 0.001). However, at 0.5%, essential oils of C. graveolens, D. ambrosioides, C. berlandieri and B. vulgaris significantly reduced the mean survival time by 55, 53 and 52 % for the last 2 plant species, as compared to the control (65.0 ± 2.6 d) (Fig. 1). The control and the stabilizer (54.9 ± 3.3 d) had similar mean survival time (Gehan-Breslow Test = 39.4, df = 6, p < 0.001). Aqueous extracts at 5% did not have an effect on mean survival time (Gehan-Breslow Test = 3.2, df = 5, p > 0.05). It ranged from 52.6 ± 3.6 d (control) to 63.5 ± 2.4 d (C. album).
Effects of essential oils and aqueous extracts of Chenopodiaceae plant species on mean (± SE) Copitarsia decolora pupal period (d).
Effects of essential oils and aqueous extracts of Chenopodiaceae plant species on mean (± SE) Copitarsia decolora fecundity. Number of mating pairs per treatment shown in parentheses.
Our results show that EOs from Chenopodiaceae plants negatively affect growth and reproduction of C. decolora. Most EOs treatments reduced larvae and pupae weight and increased the duration of the larval and pupal period compared with the control groups. Also, the inclusion of EOs into the diets reduced mean survival time and produced large larvae mortality during the first 10 days of the experiments. Most important is the reduction on fertility; this ranged from 70 to 99% with EOs at 0.5% and from 10 to 75% with EAs. Aqueous extracts also reduced fertility by 10 to 75%. The use of EOs or AEs for managing C. decolora may reduce the selection pressure on the populations by delaying or preventing the onset of insecticide resistance. In addition, low reproductive potential will further reduce the next generation of the pest.
The most promising plant was C. graveolens as its EOs at 0.1 and 0.5% affected C. decolora at various stages. Treatment with these EOs resulted in small larvae and pupae, which would reduce the potential capacity of C. decolora to produce eggs as adults. Further such treatment prolonged the larval period and thereby increased the chance of larvae to being killed by predators. Moreover such enhanced larval mortality would strongly reduce reproductive capacity of the population. Rodríguez et al. (1982) found that AEs from C. graveolens at 5% had no effect on the biology of the Noctuid Spodoptera frugiperda (Smith), however, in our study; this plant significantly reduced the reproductive potential of C. decolora. Methanolic extract of leaves and twigs of C. graveolens at 1 mg/mL showed trypanocidal activity over Trypanosoma cruzi (Zoomastigophora: Trypanosomatidae) after 48 h incubation (Abe et al. 2002), while the hexane extract of leaves and stems of C. graveolens (500 mg/L) caused a 98% mortality of Fasciola hepatica (Trematoda: Fasciolidae) after 24h (Ibarra-Moreno et al. 2012).
Effects of essential oils and aqueous extracts of Chenopodiaceae plant species on mean (± SE) Copitarsia decolora fertility.
Another promising plant is B. vulgaris as its EOs at 0.5% reduced larval weight, increase the duration of the pupal period and decimated the fecundity and fertility of C. decolora. Saponins have been suggested as responsible for the insecticidal activity of B. vulgaris because they decreased survival, reduced food intake, reduced weight gain, slowed down development and lowered reproductive potential. It is hypothesized that saponins, due to their bitterness, could either make the food less attractive to eat (repellent/deterrent activity), cause digestive problems, interrupt the molting process or have toxic effects on cells (De Geyter et al. 2007).
Beta vulgaris contains a large number of compounds such as, fatty acids (palmitic, oleic, linoleic, stearic, arachidonic), phenolic acids (syringic, caffeic, vanillic, chlorogenic), alkaloids (betanin), flavonoids (myricetin, quercetin, kaempferol) derivatives of betalains (acid betalamic, betaxatin, betacianin), derivatives of apigenin (vitexin, vitexin- 2-O-rhamnoside, vitexin-2-O-xyloside), among others. Some of these compounds have been reported as having insecticidal or repellent effects (Ninfali & Angelino 2013). However, it is difficult to attribute the reduction on reproductive potential of C. decolora to any compound without specific tests.
The essential oils of C. berlandieri at 5% successfully prolonged the larval and pupal periods and reduced fecundity and fertility > 94%. This is the first report of insecticidal activity of C. berlandieri on larvae of a noctuid. Chaires et al. (2013) reported the presence of phenols and flavonoids in raw and cooked seeds of C. berlandieri. In our experiment we used the whole plant, not only the seeds, therefore the quantities of EOs reported by Chaires et al. (2013) could be very different from those involved in our tests. Feeding deterrence of flavonoids has been reported previously by Iwashina (2003).
Kemabonta & Okogbue (2002) reported that the D. ambrosioides (C. ambrosioides) ethanolic extract at 5% applied on seeds killed 54% of adult Callosobruchus maculatus (Fabricus) (Coleoptera: Bruchidae) and reduced their oviposition rate by 72%. However, ethanolic extracts of D. ambrosioides (C. ambrosioides) failed to affect larval development and survival of the pyralid Hypsipyla grandella (Zeller) (Mancebo et al. 2000). Twenty four h after spraying tomato (Lycopersicon esculentum [Mill.]; Solanaceae) parcels with ethanolic leaf extract of C. ambrosioides at 5%, the treated parcels presented minor numbers of adults of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) (Barbosa et al. 2011) suggesting a repellent effect. Dysphania ambrosioides (C. ambrosioides) aqueous extract at 10% reduced oviposition by 98% in Plutella xylostella (L) (Lepidoptera: Plutellidae) (Medeiros et al. 2005) and killed 20% of second instar larvae of noctuid Anticarsia gemmatalis (Hübner) (Barbieri & Fiuza, 2004).
According to Tapondjou et al. (2002), the main constituents of C. ambrosioides EOs from leaves are α-terpinen (37.6%), cymol (p-cymen) (50.0%), cis-β-farnesen (1.4%), ascaridole (3.5%) and carvacrol (3.3%). Similarly, Jardim et al. (2008) reported α-terpinen (0.9%), p-cymene (2.0%), (Z)-ascaridole (61.4%), carvacrol (3.9%) and (E)-ascaridole (18.6%). The biological effect of this oil (Bakkali et al. 2008) has been attributed to ascaridole and could be responsible for the effect of this plant on C. decolora. However, Harwood et al. (1990) tested the effect of limonene and α-pinene, components of the EOs of C. ambrosioides and C. album on the noctuid Peridroma saucia (Hübner) (Lepidoptera: Noctuidae) and found that limonene at 2% reduced larval feeding, development and pupation rate.
The application of UDA-245, an essential oil extract from C. ambrosioides var. near ambrosioides, is effective in controlling the green peach aphid, Myzus persicae (Sulzer) (Homoptera: Aphididae), the Western Flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), and greenhouse whitefly, Trialeurodes vaporariorium (Westwood) (Homoptera: Aleyrodidae). UDA-245 produced no side effects on the parasitoids Encarsia formosa (Gahan) (Hymenoptera: Aphelinidae) (Chiasson et al. 2004), Orius insidiosus (Say) and Aphidius colemani (Viereck) (Bostanian et al. 2005). Based on these results, Chiasson et al. (2004) suggested that a greenhouse integrated pest management (IPM) program using a botanical such as UDA-245 could effectively control infestations of major pests present while having a negligible effect on biological control agents.
The noctuid M. configurata fed on lentils Lens culinaris cv. ‘chilean’ and C. album plants suffered great larval mortality, and a highly reduced pupal weight (Turnock 1985). Conversely, C. album failed to cause any larval and pupal weight loss, produce a shorter larval period and had no effect on larval survival. Even in a choice experiment M. configurata larvae preferred this plant over the cabbage family species, Brassica rapa (L), B. napus., B. juncea, Sinapis alba (L), and Thlaspi arvense (L) (Brassicaceae), the Compositae Cirsium arvense (L), the Fabaceae (=Leguminosae) Pisum sativum (L) and Medicago sativa (L), and the Linaceae Linum usitatissimum (L) (Dorsdall & Ulmer 2004) indicating a differential effect of C. album to different insects.
Copitarsia decolora fed as larvae with Tagetes filifolia (Lag) (Asteraceae: Compositae) essential oil at 0.01% had an 85% reduction in fecundity but similar fertility the control (Barajas 2009). In our case, the EO of B. vulgaris at 0.5% drastically reduced both parameters indicating that this plant species may performance more effectively in pest management, regardless of the concentration of the EO.
The development and use of EOs as tool for pest management requires more information about the response of the larvae to sprayed cabbage plants and on the physical contact with the EOs. Evaluation of phytotoxicity as well as development of formulation is mandatory before attempting to use EOs in the field.
We thank 2 anonymous reviewers and the Editor for constructive comments. DAVC was funded by a Master Scholarship from the Mexican National Council of Science and Technology (CONACYT). This research was funded by grant SIP-IPN 20111131, 20121139 and FOMIX-CONACYT (No. 93211). We thank Georgina Sánchez Rivera, for looking after the insect colony and help during the experimental work. The New Zealand Institute for Plant and Food Research Limited provided facilities for writing the manuscript, and Lloyd Stringer (PFR, NZ) commented, proofread and improved the English text. AJP acknowledges support from IPN and CONACYT through a Research Fellowship (Ref. 232952). Except DAVC, all authors are Comisión de Operación y Fomento de Actividades Académicas and Estímulo al Desempeño de los Investigadores fellows.